Mr Mackay is married to a fellow Tory MP
Conservative MP Andrew MacKay has quit as parliamentary aide to David Cameron over what the party said was an "unacceptable" expenses claim.
Mr Cameron has said all Tory MPs must be able to defend their allowance claims after a series of damaging allegations about MPs' claims.
Several senior Tory figures, including Michael Gove and Alan Duncan, have agreed to repay money they claimed.
Mr MacKay, MP for Bracknell, is married to fellow Tory MP Julie Kirkbride.
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To simply hand this stolen money back and expect that to be the end of the matter is totally unacceptable.
The Conservatives said Mr MacKay had voluntarily submitted his expense claims to party officials and these had revealed "an unacceptable situation that would not stand up to reasonable public scrutiny".
Mr Cameron said Mr MacKay had made "unacceptable" claims, adding: "He will go before scrutiny to determine how much of that money needs to be repaid."
It is understood that Mr MacKay and his wife claimed second homes allowances on two separate properties.
"Although Mr MacKay maintains that those arrangements were agreed by the Fees Office, he resigned this morning with immediate effect," said a party spokesman.
The party said Mr MacKay had agreed to appear before a new committee, announced by Mr Cameron on Tuesday, set up to scrutinise MP's expense claims.
It said the panel would discuss "how much of the allowance should be paid back".
The BBC's political correspondent Reeta Chakrabarti said Mr MacKay was paying a heavy price for the public backlash against MPs' behaviour over their expenses and the strong action party leaders were now taking over the issue.
Former Tory chairman Lord Tebbit said Mr MacKay was a "good guy" but said he had done the "right thing" in stepping down given the nature of the situation.
"It ain't right," he said of the claims details. "I am glad he has recognised it is so."
Tories are complaining to me about what one calls "summary mob justice" in which all are judged guilty so that the good are punished while the real bad guys escape lightly
It comes as Westminster is rocked by the seventh day of revelations about MPs' expenses in the Daily Telegraph.
Prime Minister Gordon Brown is said to be "very concerned" about former minister Elliott Morley claiming £16,000 for a mortgage he had already paid off.
Mr Morley has said it was a "mistake" and has paid the money back but Mr Brown is holding talks with Labour's chief whip about his future.
The newspaper also alleges Mr Morley rented out a London flat designated as his main residence to another Labour MP, Ian Cawsey, a close friend and former special adviser, who named the property as his second home, allowing him to claim £1,000 a month in rent, which he paid to Mr Morley.
In November 2007, the newspaper claims, Mr Morley "flipped" his designated second home from his Scunthorpe property to his London home and for four months the two men claimed expenses on the same property.
The Commons fees office stopped the arrangement. Mr Cawsey said he had not been aware of Mr Morley's financial arrangements.